A Novel RNA Polymerase II C-terminal Domain Phosphatase That Preferentially Dephosphorylates Serine 5

Yeo, Michele; Lin, Patrick S.; Dahmus, Michael E.; Gill, Gordon N.

doi:10.1074/jbc.m301791200

Cited by 190 publications

(213 citation statements)

References 56 publications

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“…Interestingly, FCP1 is not efficiently inactivated by the phosphatase inhibitors used in our extracts (NaF and Na 3 VO 4 ). More complete inactivation of FCP1 requires EDTA and EGTA (59), which are absent from our buffers due to the requirement for divalent cations in some BRCA1 structures (60,61). Since this IIA-like band represents a minor percentage of the total p220 present in BRCA1 complexes, we have not been able to fully document its origin, although this remains an interest.…”

Section: Fig 3 Minimally Phosphorylated Brca1 Proteins Interact Prementioning

confidence: 68%

BRCA1 Associates with Processive RNA Polymerase II

Krum¹,

Miranda²,

Lin³

et al. 2003

Journal of Biological Chemistry

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The human BRCA1 tumor suppressor interacts with transcriptional machinery, including RNA polymerase II (RNA pol II). We demonstrated that interaction with RNA pol II is a conserved feature of BRCA1 proteins from several species. We found that full-length BRCA1 proteins universally fail to activate transcription in classic GAL4-UAS one-hybrid assays and that the activity associated with the human BRCA1 C terminus was poorly conserved in closely related homologs of the gene. Fractionation studies demonstrated that BRCA1 proteins from all species tested interacted specifically with hyperphosphorylated pol II (IIO), in preference to hypophosphorylated RNA pol II (IIA) expected at promoters. BRCA1-RNA pol II complexes showed evidence of a multiply phosphorylated heptad repeat domain in the catalytic subunit (p220) of RNA pol II, and the complex was highly functional in transcriptional run-off assays. Interestingly, endogenous BRCA1 associated with a large fraction of the processive RNA pol II activity present in undamaged cells, and the interaction was disrupted by DNA-damaging agents. Preferential interaction with processive RNA pol II in undamaged cells places BRCA1 in position to link late events in transcription with repair processes in eukaryotic cells.Mutations in the BRCA1 tumor suppressor gene are associated with an increased risk of breast and ovarian cancer and an elevated incidence of certain other cancers (1-3). Genetic and biochemical data place BRCA1 as a downstream target of ATM/ ATR in cellular responses to genotoxic stress, and the BRCA1 protein has been implicated in chromatin remodeling and homologous recombination functions in mitotic and meiotic cells (4 -8). BRCA1 complexes contain E3 ubiquitin-ligase activity, but the precise target(s) remains an avenue of active study (9, 10). Although BRCA1 proteins show surprisingly low sequence identity, human BRCA1 can replace the mouse gene in genetically engineered animals (11, 12), a result that implies general conservation of important functional motifs. Comparative functional studies are thus likely to play an important role in identifying critical targets of BRCA1 in human cells.A distinct literature has evolved linking BRCA1 to roles in transcription. BRCA1 protein has been shown to associate with RNA polymerase II (RNA pol II) 1 and various transcriptional regulators (13). Early on, several groups demonstrated that the C-terminal domain (CTD; amino acids 1380 -1863) of human BRCA1 scored positively in transcriptional activator trap experiments using various forms of the so-called "one-hybrid" assay (14 -18). While not elucidating a specific function, ectopic expression of full-length human BRCA1 was then shown to increase expression of stress-responsive genes including p21 (19), GADD45 (20), and p27 (21) and decreased expression of other genes, including c-Myc-regulated genes (22) and certain estrogen-regulated genes (23). However, it remains an open question whether endogenous BRCA1 directly mediates recruitment of RNA pol II to promoters of these...

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Section: Fig 3 Minimally Phosphorylated Brca1 Proteins Interact Prementioning

confidence: 68%

BRCA1 Associates with Processive RNA Polymerase II

Krum¹,

Miranda²,

Lin³

et al. 2003

Journal of Biological Chemistry

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“…In contrast, REST has been detected, together with a phosphatase, CTDSP1, on neuronal gene chromatin (33). CTDSP1 was first identified as a phosphatase for the C-terminal domain of RNA polymerase II in vitro (34). The expression of CTDSP1 decreases dramatically with differentiation of stem cells to mature neurons (33), and knockdown of CTDSP1 in a neural progenitor cell line accelerates neuronal differentiation (22).…”

Section: Discussionmentioning

confidence: 99%

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

Nesti

Corson

McCleskey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The repressor element 1 (RE1) silencing transcription factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C terminus of REST that require activity of beta-transducin repeat containing E3 ubiquitin protein ligase, βTrCP. We identify a proline-directed phosphorylation motif, at serines 861/864 upstream of these sites, which is a substrate for the peptidylprolyl cis/trans isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-terminal domain small phosphatase 1 (CTDSP1), which is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864, and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down-regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells and that ERK-dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors.T he repressor element 1 (RE1) silencing transcription factor (REST) is a transcriptional repressor that suppresses neuronal gene expression in nonneural cells, such as fibroblasts, as well as in neural progenitors (1-3). Its targets represent genes required for the terminally differentiated neuronal cell phenotype, including genes encoding voltage and ligand-dependent ion channels, receptors, growth factors, and axonal-guidance proteins (4-7). Thus, during neurogenesis, REST is progressively down-regulated to allow elaboration of the mature neuronal phenotype (3). Nonetheless, precisely how REST itself is regulated still remains an open question. Relatively little is known about either its transcriptional or posttranscriptional regulation (3,8,9). In contrast, several studies have focused on posttranslational regulation of REST (3, 10, 11), but the identity of the signaling molecules involved has received little attention.In neural progenitors and human embryonic kidney (HEK) cells, rapid REST turnover is mediated by targeting to a proteasomal pathway (3, 10, 11). REST degradation during neuronal differentiation in culture requires interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (βTrCP) for targeting to the proteasome (11). βTrCP was also required for cell-cycle-dependent degradation of REST in HEK cells (10). Two adjacent phosphorylated peptides in the C-terminal domain of REST were identified as βTrCP substrates in these studies, and function as degrons. One kinase responsible for the phosphorylation and degron activity in hippocampus is casein kinase 1, CK1 (12), but whether it function...

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“…Furthermore, phosphatase-deficient EYA3 did not interact with RNAPII in vitro (Li et al, 2003), which may facilitate degradation of RNAPII and, therefore, transcriptional silencing as well. Similarly, mutant SCP1 (small CTD phosphatase; CTD, C-terminal domain of RNAPII) competes with the integration of endogenous SCP1 in a RNAPII complex, which could lead to an increase of serine 5 phosphorylation and, thereby, increase the efficiency of cap formation and transcription (Yeo et al, 2003). Moreover, dephosphorylation of RNAPII at serine 5 by SCP1 represses transcription, whereas a phosphatase mutant of SCP1 (SCP1 PTPϪ ) activates it, just like EYA missing the phosphatase domain in a Gal4DBD transactivation assay (Silver et al, 2003;Yeo et al, 2003).…”

Section: Overexpression Of Xeya3 Phosphatase-dead Mutant Formsmentioning

confidence: 99%